title: Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy.
abstract: BACKGROUND
  Human α1-proteinase inhibitor (α1-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α1-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern.
  
  
  METHODS
  Five variants of plant-expressed recombinant α1-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α1-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy.
  
  
  RESULTS
  Urea-induced unfolding of recombinant α1-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4M to 4.3M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~5-fold at 54°C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α1-PI but provided enhanced resistance to oxidative inactivation.
  
  
  CONCLUSIONS
  Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α1-PI protein, without inflicting the inhibitory activity of the protein.
  
  
  CONCLUSIONS
  Our results demonstrate the significance of engineered modifications in plant-derived recombinant α1-PI protein molecule for further therapeutic development.
authors.affiliation: Plant Transgenic Lab, MB and GE Division, CSIR-National Botanical Research Institute, P.O. Box 436, Rana Pratap Marg, Lucknow 226 001, India. Electronic address: sj.bo@jnvu.edu.in.
authors.forename: Shweta
authors.initials: S
authors.lastname: Jha
authors.forename: Indraneel
authors.initials: I
authors.lastname: Sanyal
authors.forename: D V
authors.initials: DV
authors.lastname: Amla
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D024202:Electrophoretic Mobility Shift Assay; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase
keywords: C(m); CD; DAC-ELISA; Human α(1)-proteinase inhibitor; Oxidation resistance; PPE; Protein stability; RCL; Recombinant therapeutic protein; Single amino acid substitution; TSP; TUG; Transgenic plant; circular dichroism; direct antigen coating-enzyme linked immunosorbent assay; mid-point of transition; porcine pancreatic elastase; reactive center loop; total soluble protein; transverse urea gradient gel electrophoresis; α(1)-PI; α(1)-proteinase inhibitor